A new species of Ophryotrocha (Annelida, Eunicida, Dorvilleidae) from hydrothermal vents on the Southwest Indian Ridge

Abstract Dorvilleids were collected from hydrothermal vents on the Southwest Indian Ridge by manned submersible Jiaolong. These represent a new species of Ophryotrocha that is here described as Ophryotrocha jiaolongi sp. n. This is the first dorvilleid described from vents on the Southwest Indian Ridge. It most closely resembles another vent species, Ophryotrocha akessoni Blake, 1985 from the Galapagos Rift, but can be distinguished by its antennae, palps, jaw structure. The new species has particularly distinctive mandibles, which allow it to be easily identified.

Ophryotrocha has previously been reported from vent fields on the Central Indian Ridge (Van Dover et al. 2001, Watanabe andBeedessee 2015) and the Southwest Indian Ridge (Copley et al. 2015). However, they have not been described. In this paper, dorvilleid worms from vents field on the Southwest Indian Ridge were studied and named as the sixth Ophryotrocha species from the hydrothermal vents.

Sample collection and morphological analyses
In January 2015, the China Ocean Mineral Resource R&D Association (COMRA) cruise DY35 was carried out by the research vessel Xiangyanghong 9, visiting the Southwest Indian Ocean. Sampling from the vents field was undertaken by the manned submersible Jiaolong. Specimens collected from two sites of the Longqi vent field, were sieved through a 250 μm mesh sieve, sorted, and preserved in 95% ethanol on board. The holotype and most paratypes are deposited in the repository of the Second Institute of Oceanography (RSIO), Hangzhou, China; additional paratypes are deposited in the Scripps Institution of Oceanography Benthic Invertebrate Collection (SIO-BIC), La Jolla California, USA.
Specimens were examined and photographed using a Zeiss V20 stereomicroscope with AxioCam ICc5 camera and a Leica DM5000 compound microscope. Jaws and chaetae were analyzed by scanning electron microscope (SEM). Jaws from both holotype and paratype were obtained after digesting anterior decapitated ends with a proteinase K solution at room temperature. Once the tissue was digested, the jaw elements were cleaned with distilled water and transferred to a glass cover slip. All elements for SEM were mounted on stubs and sputter coated with platinum-palladium and imaged using a Hitachi TM1000 scanning electron microscope.
DNA extraction was done with DNeasy blood and tissue kit (Qiagen, CA, USA) following the protocol supplied by the manufacturer. About 680 bp of CO1, 500 bp of 16S and 350 bp of H3 were amplified using primers LCO1490 and CO-E (Folmer et al. 1994, Bely andWray, 2004) for CO1, 16SarL and 16SbrH (Palumbi 1996) for 16S and H3F and H3R (Colgan et al. 2000) for H3. PCR mixtures contained ddH 2 O, 1μl each primer (10 μM), 2 μl template DNA, 0.5 U of Taq polymerase (TAKARA, China), 2.5 μl of buffer solution (supplied by the polymerase manufacturer) and 0.5 μl of 2.5 mM dNTPs solution in a mixture of total 25μl. The temperature profile was as follows: 96°C/240s -(94°C/30s -50°C/30s -72°C/60s) * 35cycles -72°C/420s. PCR products were purified with QIAquick PCR purification kit (Qiagen, CA, USA) following the protocol supplied by the manufacturer. Sequencing was performed by Sangon Biotech (Shanghai, China) on an ABI 3730XL DNA analyser (Applied Biosystems). Alignments of the three genes (CO1, 16S, H3) were performed using the program MAFFT (Katoh and Standley 2013) with all DNA data of dorvilleids available from Genbank. A maximum likelihood (ML) analysis was conducted by RAxML (Stamatakis 2014) using combined data of the three genes. Description. In life, body translucent (Fig. 1a), becoming opaque white after preservation (Fig. 2a, b). Body shape elongated, slightly dorsoventrally compressed, length up to 10 mm for more than 50 chaetigers, width 1.1 mm, uniform throughout the body, slightly tapering posteriorly (Fig. 2a, b). Prostomium wider than long, anterior margin rounded, posterior medial area slightly raised. Paired antennae short, digitiform, inserted dorsally, reaching to the anteriorly rounded edge of the prostomium (Fig. 2c, Fig. 4a). Paired palps digitiform, similar length as antennae, inserted ventrallaterally on prostomium (Fig. 2d). Eyes not visible. Peristomium with two rings subequal in length to following segments, the first ring with two notches ventrally on both sides of the jaw (Fig. 2d). Complete ciliary bands are observed on peristomium segments and chaetigers. Pygidium with terminal anus, two digitiform pygidial cirri inserted laterally, similar in length with the parapodia on the last chaetigers, a small median papilla ventrally placed (Fig. 2a, b, Fig. 4b).
Etymology. Ophryotrocha jiaolongi sp. n. is named after the Chinese manned submersible Jiaolong, in recognition of its successful expedition to the hydrothermal vents of the Southwest Indian Ridge.  1 mm (a, b), 0.5 mm (c, d).
Remarks. The complex pharyngeal jaw apparatus, which is morphologically well characterized by the presence of ventral mandibles and dorsal maxillae, is an important diagnostic feature in Dorvilleidae (Rouse and Pleijel 2001). Mandibles of most Ophryotrocha species have been reported with a distally serrated edge or smooth anterior margin with anterior mandibular peaks. Ophryotrocha jiaolongi sp. n. has distinctive mandibles, with folded sub-triangular cutting plates, a distally smooth edge and a single anterior blunt peak, which easily distinguish it from other Ophryotrocha species. Among   Bootstrap support values (only higher than 50 were shown) were generated with a rapid bootstrapping algorithm for 1000 replicates. Double asterisk indicates support value of 100, single asterisk indicates support value of 95 or above.
Ophryotrocha species, O. jiaolongi sp. n. most closely resembles O. akessoni Blake, 1985, in the general morphology of the prostomium, peristomium, ciliary bands, parapodia and chaetae, as well as in mandibular and maxillary structure. Ophryotrocha jiaolongi sp. n. differs from O. akessoni in having shorter antennae and palps and slight differences in jaw structure. The maxillae appear to be P-type in both species, although Blake referred to that of O. akessoni as tending towards K-type in the adult. Ophryotrocha jiaolongi has alternating large and small teeth on D4-D5, while O. akessoni has alternating large and small teeth on the forceps and D1.
DNA. Sequences of Ophryotrocha jiaolongi sp. n. are deposited at NCBI Genbank with accession numbers CO1 KY906961-KY906965, 16S MF398963-MF398967, and H3 MF398968-MF398972. Preliminary phylogenetic analysis of the DNA data suggests that O. jiaolongi sp. n. is closely related to O. clava from whale bones. However, only one sequence of vent species (O. globopalpata) is currently available, which is located in a different clade from the new species. Further DNA data is being acquired from other vent Ophryotrocha species, which will help us to get a better understanding of the relationship among vents Ophryotrocha species in the near future (Zhang et al. in prep.).